Project Summary Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor involved in various cellular biochemical reactions and contributes to the regulation of Ca2+ signaling, chromatin structure, DNA repair and lifespan. To date, how eukaryote cells transport and utilize NAD+ precursors as well as the signaling pathways that regulate NAD+ homeostasis remain unclear. Studying NAD+ homeostasis is also complicated by the complex and dynamic flexibility of precursors cells use to generate NAD+. Nicotinamide riboside (NR) and nicotinamide (NAM) are key pyridine metabolites that play important roles in maintaining NAD+ pools as well as calorie restriction (CR)-induced lifespan. To further understand the mechanisms of NAD+ homeostasis, we have established NR-specific and NAM-specific reporter genetic screen systems and identified yeast mutants with altered NAD+ metabolism. Our studies suggest that phosphate responsive PHO signaling and glucose signaling pathways contribute to controlling the NR branch of NAD+ metabolism. We have recently identified additional novel NAD+ homeostasis factors in the NAM branch. The current proposal builds on our recent studies of these factors and the interplay between components in NR/NAM/NAD+ metabolism and longevity-related nutrient signaling pathways. The long-term goal is to understand the mechanisms by which cells maintain NAD+ homeostasis in response to changes in growth conditions. The major hypothesis is that NAD+ homeostasis is modulated by nutrient-sensing signaling pathways, which play important roles in determining cell fitness and survival. Intracellular compartmentalization of NAD+ intermediates and homeostasis factors also contribute to the complex interplay of NAD+ homeostasis factors and nutrient sensing pathways. The specific aims of the projects are: Aim 1) To study how NAD+ precursor assimilation factors regulate NAD+ homeostasis, Aim 2) To study the cross-regulation of NAD+ homeostasis and nutrient sensing pathways, and Aim 3) To Identify and study novel NAD+ homeostasis factors in the NA/NAM salvage pathway. To achieve theses goals we will employ a combination of molecular genetics and biochemical methods to analyze genes, proteins and pathways involved. These studies will increase our understanding of how eukaryotic cells regulate NAD+ homeostasis in response to changes in growth conditions, and which longevity-related nutrient sensing signaling pathways are involved. Overall Significance: Our findings will contribute to the understanding of the molecular basis of the complex regulation of NAD+ homeostasis and cell life span as well as metabolic disorders related to aberrant NAD+ metabolism in human.